背景:早产儿慢性肺病,称为支气管肺发育不良(BPD),缺乏有效的治疗方法,强调需要反映人类病理学的临床前测试系统来识别因果途径和测试新化合物。源自人类多能干细胞(hPSC)的肺泡类器官是研究BPD等远端气道疾病的有前途的测试平台。但是目前的方案不能准确复制天然肺的远端小生境环境。在这里,我们研究了肺泡的细胞成分和胎儿呼吸运动对hPSC衍生的肺泡类器官形成的贡献。
方法:人PSC在2D培养物中分化成肺祖细胞(LPC),然后在去除共发育的中胚层细胞之前和之后进一步分化成肺泡类器官。LPC也在有和没有人胎儿肺成纤维细胞的Transwell®共培养物中分化。使用Flexcell®系统使形成的类器官经受阶段性机械应变。通过流式细胞术评估类器官和Transwell®培养物中的分化,免疫荧光,和qPCR的肺上皮和肺泡分化标志物,包括GATA结合蛋白6(GATA6),E-钙黏着蛋白(CDH1),NK2Homeobox1(NKX2-1),HT2-280、表面活性蛋白B(SFTPB)和C(SFTPC)。
结果:我们观察到共同发育的间充质祖细胞促进hPSC衍生的肺类器官内的肺泡2型上皮细胞(AEC2)分化。通过共培养hPSC-NKX2-1+肺祖细胞与人胚肺成纤维细胞证实了这种对AEC2分化的间质效应。刺激作用不需要成纤维细胞和NKX2-1+肺祖细胞之间的直接接触。此外,我们证明了hPSC衍生的肺类器官的阵发性机械变形,模仿原位胎儿呼吸运动,AEC2分化增加而不影响近端上皮分化。
结论:我们的数据表明,生物物理和间充质成分在体外促进hPSC衍生的远端类器官内的AEC2分化。
BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.
METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC).
RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation.
CONCLUSIONS: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.